Effect of sulfonated lignin on enzymatic activity of the ligninolytic enzymes Cα-dehydrogenase LigD and β-etherase LigF

NAD⁺-dependent Cα-dehydrogenase LigD and glutathione-dependent β-etherase LigF which selectively cleave the β-O-4 aryl ether linkage present in lignin, are key-enzymes for the biocatalytic depolymerization of lignin. However, the catalytic efficiency of the two enzymes is low when they are used to break down the β-aryl ether linkage in natural lignin. When sulfonated lignin was added to LigF hydrolysis reactions, the conversion rate of MPHPV decreased significantly from 99.5% to 32.6%. On the contrary, sulfonated lignin has little affection on LigD, which the conversion rate of GGE only decreased from 41.7% to 41%. The strong nonspecific interactions of enzymes onto sulfonated lignin detected by surface plasmon resonance (SPR) and isothermal titration calorimetric (ITC) was obvious and universal, which can reduce enzyme activity of many enzymes, including ligninolytic enzyme β-etherase LigF. To elucidate the exact mechanisms by which β-etherase LigF interact with lignin, molecular modeling was applied. Finally, analysis on catalytic efficiency of LigD and LigF in different concentrations and molecular weights of sulfonated lignin, solution ionic strength, pH, temperature and concentration of Tween 80 revealed that electrostatic interactions and hydrophobic interactions play important roles in absorption between LigF and sulfonated lignin.     

Rewired phenolic metabolism and improved saccharification efficiency of a Zea mays cinnamyl alcohol dehydrogenase 2 (zmcad2) mutant

Lignocellulosic biomass is an abundant byproduct from cereal crops that can potentially be valorized as a feedstock to produce biomaterials. Zea mays CINNAMYL ALCOHOL DEHYDROGENASE 2 (ZmCAD2) is involved in lignification, and is a promising target to improve the cellulose-to-glucose conversion of maize stover. Here, we analyzed a field-grown zmcad2 Mutator transposon insertional mutant. Zmcad2 mutant plants had an 18% lower Klason lignin content, whereas their cellulose content was similar to that of control lines. The lignin in zmcad2 mutants contained increased levels of hydroxycinnamaldehydes, i.e. the substrates of ZmCAD2, ferulic acid and tricin. Ferulates decorating hemicelluloses were not altered. Phenolic profiling further revealed that hydroxycinnamaldehydes are partly converted into (dihydro)ferulic acid and sinapic acid and their derivatives in zmcad2 mutants. Syringyl lactic acid hexoside, a metabolic sink in CAD-deficient dicot trees, appeared not to be a sink in zmcad2 maize. The enzymatic cellulose-to-glucose conversion efficiency was determined after 10 different thermochemical pre-treatments. Zmcad2 yielded significantly higher conversions compared with controls for almost every pre-treatment. However, the relative increase in glucose yields after alkaline pre-treatment was not higher than the relative increase when no pre-treatment was applied, suggesting that the positive effect of the incorporation of hydroxycinnamaldehydes was leveled off by the negative effect of reduced p-coumarate levels in the cell wall. Taken together, our results reveal how phenolic metabolism is affected in CAD-deficient maize, and further support mutating CAD genes in cereal crops as a promising strategy to improve lignocellulosic biomass for sugar-platform biorefineries.     

Impact of protein blocking on enzymatic saccharification of bagasse from sugarcane clones

Lignin plays an important functional and structural role in plants, but also contributes to the recalcitrance of lignocellulosic biomass to hydrolysis. This study addresses the influence of lignin in hydrolysis of sugarcane bagasse from conventional bred lines (UFV260 and UFV204) that were selected from 432 field-grown clones. In addition to higher sugar production, bagasse clone UFV204 had a small, but statistically significant, lower insoluble lignin content compared with clone UFV260 (15.5% vs, 16.6%) and also exhibited a significantly higher cellulose conversion to glucose (81.3% vs. 63.3%) at a cellulase loading of 5 (filter paper unit) FPU/g of glucan or 3 FPU/g total solids for liquid hot water pretreated bagasse (200°C, 10 min). The enzyme loading was further decreased by 50% to 2.5 FPU/g glucan and resulted in a similar glucan conversion (88.5%) for clone UFV204 when the bagasse was preincubated with bovine serum albumin at pH 4.8 and nonproductive binding of cellulase components was blocked. Comparison of Langmuir adsorption isotherms and differential adsorption of the three major cellulolytic enzyme components endoglucanase, cellobiohydrolase, and β-glucosidase help to explain differences due to lignin content.     

Recent advances in fungal cellobiose oxidoreductases

When grown on cellulose, the white-rot fungus Phanerochaete chrysosporium (Sporotrichum pulverulentum), produces two cellobiose oxidoreductases, i.e., cellobiose:quinone oxidoreductase (CBQ) and cellobiose oxidase (CBO). Similar cellobiose-oxidizing enzymes, capable of utilizing a wide variety of electron acceptors, have been detected in many other fungi. However, the role of the cellobiose oxidoreductases in white-rot fungi, or in any fungi for that matter, is still not known. The original role ascribed to CBQ was as a link between cellulose and lignin degradation. CBQ has been shown to reduce quinones and phenoxyradicals released during lignin degradation concomitantly oxidizing cellobiose and other cellodextrins released during cellulose degradation. Thus, one function proposed for the cellobiose oxidoreductases is to prevent repolymerization of phenoxyradicals formed when phenoloxidases (peroxidases and laccases) attack lignin and lignin degradation products. However, evidence obtained so far indicates that the presence of CBO/CBQ with lignin peroxidases and laccases actually reduces the rate of oxidation of lignin degradation products. CBQ has a molecular mass of about 60 kD and contains an FAD cofactor. CBO contains both heme and FAD, and has a mass of about 90 kD. It has recently been demonstrated that CBO can be proteolytically cleaved into FAD and heme domains. The FAD domain of CBO seems to have all the properties of CBQ, suggesting that CBQ is a cleavage product of CBO. Whether CBO is a precursor of CBQ is not yet known. CBO and CBQ can be distinguished not only by the differences in their spectral properties, but also by the ability of CBO, but not CBQ, to reduce cytochrome c. Both CBO and CBQ have a cellulose-binding domain (CBD), as do a large number of endoglucanases and cellobiohydrolases. The induction-repression patterns regulating cellobiose oxidoreductase genes are not known in any detail. Most reports point to induction during cellulose degradation, but repression has not been studied. Induction has also been suggested to occur by addition of lignosulfonate to the medium.     

Effect of chemical modification of lignin on the gluebond performance of lignin-phenolic resins

Lignin obtained from eucalyptus wood by acetic acid pulping was methylolated or phenolated and used to prepare lignin-phenol-formaldehyde resins. The amount of formaldehyde consumed in the methylolation reaction, and supporting comparison of pre- and post-methylolation ¹H and ¹³C NMR spectra, showed the reactivity of the crude acetosolv lignin with formaldehyde to be relatively high. Pine and eucalyptus plywood boards manufactured using the resins prepared with the modified lignins complied with European Standard EN 314-1:1993 for WBP quality boards and gave knife test results similar to those of boards manufactured with a commercial phenol-formaldehyde resin.     

The influence of preformed defences on the dynamic wound response in Spruce bark

1. Two of the principal defences in conifer bark against attack by bark beetles and associated fungi, the flow of preformed resin and the dynamic wound response (DWR), are commonly regarded as separate, sequential responses to attack. In this paper the concentration of three preformed defences (resin, total polyphenols and lignified stone cell masses) was measured at different heights on the stem of Norway and Sitka Spruce to determine their effect on the size of lesions formed during the DWR to inoculation with three facultatively pathogenic fungi ( Phacidium coniferarum, Ophiostoma piceae and Cryptosporiopsis sp.) inoculated during the dormant season.
2. There was considerable within-tree variation in concentration of lignin and there was a dose-dependent negative effect of lignin on lesion size. The form of the relationship, however, was influenced by fungal and tree species and for some fungi, also by an individual tree effect.
3. Results suggest that resistance of conifer bark to pests and pathogens depends on an interaction between preformed and induced defences.     

Suppression of a BAHD acyltransferase decreases p-coumaroyl on arabinoxylan and improves biomass digestibility in the model grass Setaria viridis

Grass cell walls have hydroxycinnamic acids attached to arabinosyl residues of arabinoxylan (AX), and certain BAHD acyltransferases are involved in their addition. In this study, we characterized one of these BAHD genes in the cell wall of the model grass Setaria viridis. RNAi silenced lines of S. viridis (SvBAHD05) presented a decrease of up to 42% of ester-linked p-coumarate (pCA) and 50% of pCA-arabinofuranosyl, across three generations. Biomass from SvBAHD05 silenced plants exhibited up to 32% increase in biomass saccharification after acid pre-treatment, with no change in total lignin. Molecular dynamics simulations suggested that SvBAHD05 is a p-coumaroyl coenzyme A transferase (PAT) mainly involved in the addition of pCA to the arabinofuranosyl residues of AX in Setaria. Thus, our results provide evidence of p-coumaroylation of AX promoted by SvBAHD05 acyltransferase in the cell wall of the model grass S. viridis. Furthermore, SvBAHD05 is a promising biotechnological target to engineer crops for improved biomass digestibility for biofuels, biorefineries and animal feeding.     

Bacteria and lignin degradation

Lignin is both the most abundant aromatic (phenolic) polymer and the second most abundant raw material. It is degraded and modified by bacteria in the natural world, and bacteria seem to play a leading role in decomposing lignin in aquatic ecosystems. Lignin-degrading bacteria approach the polymer by mechanisms such as tunneling, erosion, and cavitation. With the advantages of immense environmental adaptability and biochemical versatility, bacteria deserve to be studied for their ligninolytic potential.     

Occurrence of Δ1(10) Gibberellin A1 Counterpart,GA1, GA4 and GA7 in Somatic Cell Embryo Cultures of Carrot and Anise

Amine oxidase of Aspergillus niger was inactivated by ethylenediamine under aerobic conditions, but not under anaerobic conditions. In the presence of ethylenediamine, the oxidized form of the enzyme did not react with phenylhydrazine under anaerobic conditions, but reacted slowly under aerobic conditions. These findings and previous study [Suzuki et al., J. Biochem. 69, 1065 (1971)] suggest that the oxidized form of the enzyme develops an inactive Schiff base between the carbonyl group of the enzyme and the amino group of ethylenediamine under aerobic conditions. The circular dichroic (CD) spectra in the near-ultraviolet region indicated that the structure around the inactive Schiff base was slightly different from that of the reduced form of the enzyme.